: The overall goal of the research proposed in this application continues to be the identification and characterization of cis- and trans-acting factors involved in the regulation of alternative splicing. To this end, the investigators will continue to use mini-gene constructs and particular sequence elements from the contractile protein genes alpha-tropomyosin and troponin T as a model system that will be analyzed using in vivo and in vitro cell-free splicing system. Three main specific aims will be pursued: 1.-To analyze and characterize the structure and mechanism of action of a splicing factor, Polypyrimidine Binding Protein (PBP), which they have recently purified and partially sequenced. This factor plays an important role in determining 3' splice strength and might be involved in the early steps of splice site commitment. 2.-To further determine the binding site of a negative splicing factor specific to smooth muscle cells. To isolate, purify, and characterize the mechanism of action of this factor that works by blocking the default splicing pattern. 3.-Using a highly selective genetic approach, to identify and characterize a positive splicing factor specific to mature smooth muscle cells which determines the regulated splicing pattern of the troponin T transcript. It is expected that the cloning and characterization of these three splicing factors will make a significant contribution to the understanding of both constitutive and alternative models of splicing.